BIOREACTOR AND METHOD OF USE OF SUCH BIOREACTOR

Abstract

A bioreactor includes a container (1) composed of at least one side wall (11), a bottom wall (12) and an opening (13) closed by a cap (14), such to identify an inner chamber. An intermediate wall (2) is inside the inner chamber, to divide the inner chamber in an upper chamber (31) and a lower chamber (32). The intermediate wall (2) has at least one hole (21). The intermediate wall (2) has at least one region resting on the bottom wall and/or the side wall and provides at the hole (21) at least one housing seat (26) for a membrane (22).

Claims

1. Bioreactor comprising a container having at least one side wall, a bottom wall and an opening closed by a cap to define an inner chamber, an intermediate wall inside said inner chamber an intermediate wall to divide said inner chamber in an upper chamber and a lower chamber, said intermediate wall having at least one hole, wherein said intermediate wall has at least one region resting on said bottom wall and/or said side wall, said intermediate wall providing at said hole at least one housing seat to house at least one membrane.

2. Bioreactor according to claim 1, wherein said intermediate wall comprises a cup element, the cup element has the membrane placed at the bottom.

3. Bioreactor according to claim 2, wherein said cup element at an upper rim has at least one protruding region resting on said side wall.

4. Bioreactor according to claim 2, wherein said housing seat is obtained in the thickness of the bottom of said cup element.

5. Bioreactor according to claim 4, comprising at least one support element perforated at the center, the support element is inserted into said housing seat, said membrane resting on said support element.

6. Bioreactor according to claim 5, comprising one or more support elements, said support elements being inserted as stacked into said housing seat and said membrane resting on said support elements.

7. Bioreactor according to claim 1, comprising at least two support elements, said two support elements being inserted into said housing seat and said membrane being interposed between said two support elements.

8. Bioreactor according to claim 1, comprising a layer of gluing material interposed between said membrane and said support element.

9. Bioreactor according to claim 2, wherein said membrane is made as one piece with said cup element.

10. Bioreactor according to claim 1, comprising means for height adjustment of said cup element.

11. Bioreactor according to claim 1, wherein said at least one side wall delimiting said upper chamber and/or said at least one side wall delimiting said lower chamber and/or said at least one cap and/or said bottom wall have at least one inlet port and at least one outlet port open for connection of corresponding hydraulic circuits or closed by a polymer septum for drawing fluid through a syringe.

12. Bioreactor according to claim 1, comprising at least one plate element with a specific thickness and shape, the plate element is placed in contact with said bottom wall to limit volume of the lower chamber.

13. Bioreactor according to claim 1, comprising at least one fluidic circuit connected to said lower chamber and/or to said upper chamber, the fluidic circuit having at least one pump or other device for moving fluids, a delivery pipe and return pipe.

14. Bioreactor according to claim 1, wherein said delivery pipe and/or said return pipe and/or said ports have at least one valve or point of access for drawing at least a part of the flowing fluid.

15. Bioreactor according to claim 1, wherein the cap has a sealing element for liquid and/or gaseous fluids.

16. Bioreactor according to claim 1, comprising one or more sensors inside or outside the lower chamber and/or upper chamber and/or walls delimiting the bioreactor.

17. Bioreactor according to claim 1, wherein said membrane is made at least partially of cells of living tissue.

18. System for detecting and analysing passage and/or absorption of cells, molecules, particles, compounds, substances, nutrients, pollutants, growth factors, cell clusters, drugs and/or matter of any kind or electromagnetic radiations, the system comprising at least one or more bioreactors made according to claim 1, the bioreactors are connected with one another through a fluidic circuit.

19. Method for analysing passage and/or absorption of cells, molecules, particles, compounds, substances, nutrients, pollutants, growth factors, cell clusters, drugs and/or matter of any kind or electromagnetic radiations, the method comprising using at least one bioreactor according to claim 1, the method comprising the following steps: a) generation of a fluid flow in the lower chamber and/or upper chamber; b) collection and analysis of a part of flowing fluid and/or of matter contained therein or of transmitted radiation and/or collecting and analysing the membrane.

20. Method according to claim 19 comprising a step of inserting biological material in the upper chamber and/or lower chamber, and a following step analysing the biological material after effect of passage of fluid and/or molecules contained therein and/or of the passed radiation.

21. Method according to claim 19, comprising a step adjusting velocity of the fluid flow.

22. Method according to claim 19, comprising a step connecting at least two bioreactors through a fluidic circuit.

23. Method according to claim 19, wherein connection of bioreactors comprises the following steps: connecting the inlet port of the lower chamber of a first bioreactor with the outlet port of the upper chamber of a second bioreactor, connecting the outlet port of the lower chamber of the first bioreactor with the inlet port of the upper chamber of the second bioreactor.

24. Method according to claim 23, comprising a step of inserting tumor tissues or cells, biopsies, or cellularized materials or cell clusters, in the upper chamber of the first bioreactor and a metastasizable target tissue in the lower chamber or upper chamber of said second bioreactor, as an ex vivo/in vitro metastasis model.

25. Method according to claim 19, comprising a step injecting one or more drugs or molecules of interest in the upper chamber and/or lower chamber of the first and/or second bioreactor, as ex vivo/in vitro disease model for research purposes, safety and efficacy studies, pharmacokinetic and pharmacodynamics studies, drug discovery, and/or drug repositioning.

26. Method according to claim 19, comprising a step injecting one or more drugs or molecules for therapeutic use in the upper chamber and/or lower chamber of the first and/or second bioreactor, for therapeutic purposes and personalized medicine purposes.

Description

[0101] These and other characteristics and advantages of the present invention will be more clear from the following description of some embodiments shown in annexed drawings wherein:

[0102] FIG. 1 is a view of a schematic diagram of the bioreactor of the present invention according to one embodiment;

[0103] FIGS. 2a and 2b are two exploded views of the bioreactor of the present invention according to a possible embodiment;

[0104] FIG. 3 is an exploded view of the bioreactor of the present invention according to a possible embodiment;

[0105] FIGS. 4a to 4d are some details of the components of the bioreactor of the present invention;

[0106] FIGS. 5a and 5b are two views of the bioreactor in the closed condition;

[0107] FIGS. 6a and 6b are two possible configurations of the connection between hydraulic circuit and bioreactor of the present invention;

[0108] FIGS. 7a and 7b are the bioreactor of the present invention according to a possible variant embodiment;

[0109] FIG. 8 is the bioreactor of the present invention in combination with a hydraulic circuit;

[0110] FIG. 9 is the system analysing the cell passage in the metastasis model of the present invention.

[0111] It is specified that the figures annexed to the present patent application describe some embodiments of the bioreactor of the present invention and are shown to better understand characteristics and advantages thereof.

[0112] Therefore such embodiments have to be intended for merely illustration purposes and not as a limitation to the inventive concept of the present invention, regarding the provision of a bioreactor with replaceable components and easy to be adapted to the different operating needs.

[0113] FIG. 1 shows a section of the bioreactor of the present invention, according to a possible embodiment.

[0114] The bioreactor 1 comprises a container 1 composed at least of a side wall 11, a bottom wall 12 and an opening 13 closed by a cap 14, such to identify an inner chamber.

[0115] Moreover there is provided an intermediate wall 2, placed inside the inner chamber, such to divide the inner chamber in an upper chamber 31 and a lower chamber 32.

[0116] The intermediate wall 2 can be made according to different embodiments, shown in FIGS. 1 to 4d.

[0117] Such embodiments share the fact that the intermediate wall has a hole 21 intended to house a membrane 22.

[0118] The membrane 22 allows cells, molecules, particles, compounds, substances, nutrients, pollutants, growth factors, cell groups (clusters), drugs and/or matter of any kind or electromagnetic radiations to pass from the upper chamber 31 to the lower chamber 32 and vice versa.

[0119] In order to carry out analyses of interest and as it will be described below, according to a possible embodiment a sample of biological material 3 is placed in contact with the membrane 22 such to analyze cells belonging to the sample 3 passing through the membrane 22 from the upper chamber 31 to the lower chamber 32.

[0120] For practical purposes the hole 21 is placed at the center of the intermediate wall 2, but it can be placed also in an eccentric manner.

[0121] Moreover, for the sake of constructional convenience, the container 1 preferably has a cylindrical shape, but it can have any shape without the need of modifying the characteristics of the components described below.

[0122] Advantageously the container 1 is composed of plastic material, preferably a biocompatible polymer easy to be sterilized in autoclave, such as polycarbonate or the like.

[0123] Preferably the inner chamber has a diameter ranging from 3 centimeters to 5 centimeters, while the side wall 11 has a thickness ranging from 0.7 to 7 millimeters, allowing an optimal compromise to be obtained between mechanical strength, lightness depending on the method used for connecting hydraulic pipes to the system, as it will be described below.

[0124] Preferably also the cap 14 and the intermediate wall 2 are composed of a biocompatible polymer material.

[0125] As mentioned above the membrane 22 can be composed of a polymer material obtained by electrospinning process.

[0126] The intermediate wall 2 is shown in FIG. 1 as horizontally arranged, but obviously it is also possible to provide it to be inclined, depending on operating needs.

[0127] With particular reference to FIG. 1 and to FIGS. 4a to 4d the intermediate wall 2 is composed of a plate element fastened to the side wall 11 of the container 1.

[0128] Particularly the intermediate wall 2 is composed of an annular element providing the hole 21 at the center.

[0129] Moreover the intermediate wall 2 has two threaded holes 23 to allow screws (not shown) to be fastened to remove the intermediate wall 2 from the container 1.

[0130] According to a possible embodiment it is possible to provide the intermediate wall 2 to be fastened for example by resting and/or gluing it to two wedges provided in the lower chamber 32 (not shown).

[0131] Such wedges can be made as one piece with the intermediate wall 2.

[0132] According to the variant shown in FIGS. 4a to 4d, the intermediate wall 2 further has a gasket of the O-ring type 24 interposed between the outer wall of the intermediate wall 2 and the inner wall of the container 1.

[0133] Such gasket is made of autoclavable and high temperature resistant material and it allows the upper chamber 31 and the lower chamber 32 to be hermetically separated.

[0134] FIGS. 4a and 4b show two side sections of the intermediate wall 2.

[0135] As seen in FIG. 3, the hole 21 is made such to obtain a shoulder intended to form a housing seat 26 where the membrane 22 is inserted.

[0136] The membrane 22 can be inserted in the housing seat and possibly it can be fastened, for example by gluing it.

[0137] As an alternative, according to the shown embodiment, the membrane 22 is inserted in the housing seat 26 by using removable fastening means composed of two support elements 25 that fasten the membrane 22 in place.

[0138] The two jaw elements 25 are preferably composed of two annular elements 25, FIGS. 4c and 4d, that are overlapped, with the membrane 22 being fitted therebetween, such that the membrane 22 can cover the hole of the annular elements 25 and can allow the lower chamber 32 and the upper chamber 31 to be in fluidic communication.

[0139] The annular elements 25 can be fastened to the intermediate wall 2 in any of the manners known in prior art.

[0140] The annular elements 25 can be further simply placed on the intermediate wall 2, inside the housing seat 26, or can be fastened by means of specific glues.

[0141] According to a possible embodiment the annular elements can be made of silicone.

[0142] It is possible to provide any number of annular elements 25 and arranged on the basis of operating needs.

[0143] As said above, it is possible to provide only one annular support element 25, inserted in the housing seat 26 on which the membrane 22 is placed: in this case the membrane can be simply put on or even glued to the annular support element 25.

[0144] As an alternative, such as shown in FIG. 4a, it is possible to provide at least two annular support elements 25 between which the membrane 22 is interposed, a sandwich arrangement that is then inserted into the housing seat 26.

[0145] According to a possible embodiment it is possible to provide a layer of gluing material interposed between the membrane 22 and one and/or both the annular support elements 25.

[0146] According to a further embodiment, as an alternative or in combination with the variant just described, it is possible to provide the membrane 22 to be height adjusted by using a specific number of support elements 25, by simply stacking such support elements 25 inside the housing seat 26 till reaching the desired thickness, then the membrane 22 is rested on the last support element 25.

[0147] It is clear how the described configurations do not necessarily require the membrane to be fastened, but it is simply rested.

[0148] The remarks just mentioned related to the several configurations of the support elements 25 have been described with reference to the intermediate wall being made as a plate element, but such remarks can be provided in combination with different embodiments of the intermediate wall.

[0149] FIGS. 2a to 3 show two possible embodiments, where the intermediate wall is composed of a cup element, wherein the membrane is placed at the bottom wall of the cup.

[0150] Particularly FIGS. 2a and 2b show two exploded views of the bioreactor of the present invention, wherein the intermediate wall is composed of a cup element 210 fitted into a container 110.

[0151] The fact of fitting the cup element 210 in the container 110 divides the inner chamber of the container 110 in two chambers, a lower chamber, delimited by inner wall of the container 110 and by outer walls of the cup element 210, and a upper chamber delimited by the inner chambers of the cup element 210 and by the cap 14.

[0152] The cup element 210 has an upper protruding rim 211 that, in the fitted condition, contacts a step shoulder obtained inside the container 110 and visible in FIG. 2a.

[0153] Both the protruding rim and the cup element can provide any shape, different from the circular or cylindrical shape shown in the figure.

[0154] Moreover the protruding rim can also be discontinuous, that is not provided all along the diameter of the cup element, by providing protruding rim portions allowing the function thereof to be accomplished.

[0155] Therefore in the fitted condition the passage from the upper chamber to the lower chamber and vice versa is possible only through the membrane 22, that can be housed at the bottom of the cup element 210.

[0156] As an alternative it is possible to provide the membrane 22 to be made as one piece with the cup element 210.

[0157] With reference to FIG. 3, the bioreactor has a cup element 200 where the housing seat 26 for the membrane 22 is formed in the thickness of the bottom.

[0158] Inside the housing seat 26 it is possible to provide one or more annular support elements 25, according to configurations described above.

[0159] Moreover the cup element 200 has some appendages 201 at the upper rim, for example but not necessarily three appendages.

[0160] With the cup element 200 in the condition fitted into the container 100, such appendages 201 are in contact with the seat 101 obtained in the thickness of the side walls of the container 100.

[0161] Moreover the bottom of the cup element 200 in the fitted condition is in contact with the step 102, obtained on the side walls of the container 100.

[0162] Therefore advantageously the outer diameter of the cup element 200 corresponds to the inner diameter of the container 100, such that the lower chamber of the bioreactor is delimited below by the bottom wall of the container 100 and at the top by the bottom wall of the cup element 200, while the upper chamber is delimited by the inner surfaces of the cup element 200 and by the cap 14.

[0163] Regardless of the realization of the cup element, it is possible to provide means for height adjusting it by using for example shims cooperating with the rim 211 or with appendages 201.

[0164] FIGS. 5a and 5b show two views of the bioreactor in the closed condition, according to variant embodiments of FIGS. 2a-2b and 3 respectively.

[0165] FIGS. 7a and 7b show a further variant embodiment of the bioreactor of the present invention, according to which it is possible to provide at least one plate element 4, insertable within the lower chamber 32 to adjust the volume thereof.

[0166] The plate element 4 can have any size and shape, suffice that is serves the function of occupying part of the volume of the lower chamber 32.

[0167] According to the embodiment shown in FIGS. 7a and 7b the plate element 4 has a specific thickness and a shape corresponding to the bottom wall 12, such that different plate elements 4 can be stacked on each other, such as shown in FIG. 7b.

[0168] The characteristics of the bioreactor described above, allow the bioreactor to have a high adaptability to operating needs, therefore it is clear that it is possible to reduce the volume of the lower chamber not only by means of the plate elements, but also by modifying the shape of the lower chamber.

[0169] With reference again to FIGS. 1 to 3, it is possible to note how the side wall 11 delimiting the upper chamber 31 and the side wall 11 delimiting the lower chamber 32 have at least one inlet port 15, 16 and at least one outlet port 17, 18 for the connection of corresponding hydraulic circuits.

[0170] Inlet ports 15, 16 and outlet ports 17, 18 can be placed in any manner and position along the side wall 11 or lower wall 12.

[0171] In the particular case of FIGS. 7a and 7b it is clear how it is better to place the ports 16 and 18 for the lower chamber 32 near the intermediate wall, to prevent that the positioning of plate elements 4 may interfere with the fluid flow.

[0172] As an alternative it is possible to provide the inlet port 15 and outlet port 17 of the upper chamber 31 to be inserted on the cap 14, such as shown in FIGS. 2a, 2b and 3.

[0173] FIGS. 6a and 6b schematically show a possible configuration of the hydraulic circuits connected to inlet and outlet ports.

[0174] Particularly there is provided a pipe 151 and a pipe 161 connected to inlet ports 15 and 16 of the upper chamber 31 and lower chamber 32 respectively and a pipe 171 and a pipe 181 connected to outlet ports 17 and 18 of the upper chamber 31 and lower chamber 32 respectively.

[0175] In FIG. 6a the pipes 151 and 171 of the upper chamber 31 are connected to the side wall of the container 1, while in FIG. 6b the pipes 151 and 171 are provided on the cap 14.

[0176] Pipes 161 and 181 for the lower chamber 32, for constructional simplicity, are always connected to the side wall of the container 1, but they have not to be necessarily along the same axis, they can be also on different planes or axes, for example on the same plane, but with axes inclined by a specific angle.

[0177] The connection with a hydraulic circuit, some embodiments thereof being described below, can occur by closing the outlet pipe 171 of the upper chamber 31 and the inlet pipe 161 of the lower chamber 32.

[0178] Thus the hydraulic circuit will have only one delivery pipe 151, inlet of the upper chamber 31, and only one return pipe 181, outlet of the lower chamber 32, such to force the flow to pass through the interface membrane (perfusion).

[0179] FIG. 8 shows the use of the bioreactor of the present invention.

[0180] According to such embodiment there is provided a hydraulic circuit connected to the lower chamber 32.

[0181] The hydraulic circuit has a delivery pipe 51 connected to the inlet port 16, a return pipe 52 connected to the outlet port 18 and a pump 53 operating the flow of a fluid inside the circuit.

[0182] The return pipe 52 further has a three-way valve 54, but any type of valve can be provided allowing at least a part of the circulating fluid to be sampled.

[0183] A completely similar hydraulic circuit can be provided also in connection to the upper chamber 31.

[0184] With particular reference to FIG. 8, the sample 3 is placed in contact with the membrane 22, the pump 53 generates a flow flowing in the hydraulic circuit and the three-way valve 54 allows to sample a part of the flowing fluid.

[0185] On the drawn part of the fluid, some analyses can be performed, such to evaluate the concentration of cells belonging to the sample 3 to study the passage of cells through the membrane 22.

[0186] The sample 3 can be composed of an engineered material, or a biological tissue (for example biopsy) and can be cultured in the bioreactor in close contact with the membrane 22.

[0187] As set forth, the set-up with reference to FIG. 8 is particularly functional for studying diffusion of tumor cells in the body.

[0188] With the same aim it is possible to make the set-up shown in FIG. 9, intended to show the system analysing the passage of cells or molecules of biological interest (e.g. drugs) according to the present invention.

[0189] FIG. 9 shows two bioreactors connected in series, wherein the inlet ports and outlet ports of the lower chamber 32 of the bioreactor on the left are connected to the outlet and inlet ports respectively of the upper chamber 31 of the bioreactor on the right.

[0190] Moreover the inlet and outlet ports of the lower chamber 32 of the bioreactor on the right have a hydraulic circuit completely similar to that of FIG. 8.

[0191] Unlike the arrangement of FIG. 8, FIG. 9 shows also a further sample, or target organ 6 in the bioreactor on the right, that can be connected in contact with the membrane 22.

[0192] By sampling the fluid flowing through the valves 71 and 72, therefore it will be possible to evaluate not only the passage of the sample 3 through the membrane 22, but also to study the migration and adhesion of such cells to the target organ 6, as well as to study the effect of the passage of molecules of biological interest through the membrane 22.

[0193] As an alternative to the variant just described, it is possible to provide the target organ 6 to be provided in the upper chamber of the second bioreactor, such to evaluate the passage of cells of the sample 3 to the target organ 6 simply though a single barrier, represented by the membrane inserted in the first bioreactor.

[0194] As set forth, therefore it is possible by the system of FIG. 9 to evaluate the growth of a tumor in vitro.

[0195] Moreover by using the characteristics of the bioreactor of the present invention, it is possible for example to provide to insert drugs inside the hydraulic circuit to carry out preventive analyses for treating tumors grown in vitro, or other types of analyses or studies on tumors.

[0196] Finally a particularly important application of the bioreactor of the present invention is related to the use of membranes intended to simulate the skin.

[0197] In this case it is possible to evaluate how much toxic agents, pollutants, various particles or molecules, drugs or active principles or radiations (e.g. UV radiations) are able to pass through the skin and to pass in surrounding tissues and when the use of possible drugs or cosmetic solutions (for example lotions) modify such passage.

[0198] In order to carry out such method it is possible to create a fluid flow containing particles or molecules under examination (for example pollutants, or drugs or other molecules) in the upper chamber and flow of clean fluid in the lower chamber.

[0199] The membrane can be composed of human or animal skin or natural or artificial tissues, intended to simulate the human skin.

[0200] Finally cosmetic or dermatologic solutions are used placed on the membrane and, on the basis of their different types, the transmission of pollutants and/or of external agents from the upper chamber to the lower chamber is evaluated.

[0201] A possible use of the bioreactor of the present invention involving the study of diffusion of substances through the skin, but not limited to cosmetics, but related to pharmacology, for example is the analysis of the amount of lotions, creams or the like that, once spread on the skin, pass at systemic level and is absorbed by the organism.

[0202] Finally it is clear how the bioreactor of the present invention allows different applications to be obtained besides the ones described herein.

[0203] For example it is possible to use the bioreactor with epithelial tissues different from skin, such as lung barrier, gastrointestinal barrier, cornea, urethra or other epithelial tissues and not epithelial tissues.

[0204] In the case of the lung barrier it is possible to use the bioreactor to evaluate how much an external agent spreads into the organism passing though the lung barrier.

[0205] As regards gastrointestinal barrier, the bioreactor of the present invention is widely used in nutraceutical field, making it possible to analyse how much a substance can be absorbed by the organism and which amounts.

[0206] Another use, still with reference to the passage of specific substances through the gastrointestinal barrier, may be related to digestibility of such substances.